Tnf α

F4/80 (#SAB5500103) was obtained from Sigma-Aldrich (St. Louis, MO, USA). The TLR4 inhibitor TAK-242 (#614316) was obtained from Calbiochem (San Diego, CA, USA). The PI3K inhibitor wortmannin (#HY-10197) was obtained from MedChemExpress (New Jersey, USA). Collagen II (#28459-1-AP) was obtained from Proteintech Group (Wuhan, China). TLR4 (#AF7017), CD68 (#DF7518), PI3K (#DF6069) antibodies were obtained from Affinity Biosciences (Cincinnati, OH, USA). SP-D (#ab220422), MD-2 (#ab24182), TNF-α (#ab66579), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#ab181602), MMP-13 (#ab84594), and NLRP3 (ab263899) antibodies were obtained from Abcam (Cambridge, UK). Antibodies for NF-κB p65 (#8242), phospho-p65 (p-p65) (#3033), p-Akt (#4060) and Akt (#9272) were procured from Cell Signaling Technology (Boston, MA, USA). Recombinant human SP-D (rhSP-D) (#CSB-YP021175HU) was purchased from Huamei Biotech Co., Ltd (Wuhan, China). ELISA Kits of rat IL-1β (#ELK1272) and TNF-α (#ELK1396) were purchased from ELK Biotechnology (Wuhan, China). SNP (#1008) was purchased from Youcare Pharmaceutical Group Co., Ltd (Beijing, China). All of the other chemicals and reagents were of analytical grade.

The cell-free supernatants were collected from cell cultures after centrifugation at 1500 rpm for 4 minutes at 4°C and assayed for targeted proteins. The cultured cells were digested and centrifuged at 800 rpm for 4 minutes at 4°C to collect the cell precipitate. Afterwards, the cells were lysed using RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific) in the presence of protease inhibitors (Roche) and then were sent for detection of protein levels of specific cytokines by enzyme-linked immunosorbent assay (ELISA) kits. Four corneas from the same group were extracted and homogenized in 0.01 M sodium phosphate buffer (pH 9.5) containing protease/phosphatase inhibitor cocktail (1:100, Sigma) on ice. Commercially available ELISA kits were used following the manufacturer's protocols to measure the protein levels of NLRP3, IL-1β, IL-10, Areg, TNF-α, TGF-β, and VEGF-A (ELK Biotechnology). Each sample was assayed in three technical replicates. Data represented mean ± SEM of three independent experiments, and each time point of one group consisted of four mice.